The Effect Of SiRNA Interference NOX4 Gene For High Glucose Stimulate Mouse Podocytes To Produce Microvesicles

Objective:In this study, we further explore the effects of NADPH oxidase NOX4 mediated oxidative stress on the process of high glucose stimulating podocytes injury and producing microvesicles.Methods:MPC-5 was cultured in vitro, 30 mM glucose as the processing factors of high glucose(HG)group.1.Used RT-qPCR, Western Blot to detect the levels of NOX4 mRNA and protein after HG processed 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, to determine the peak point of time.2.FAM-siRNA transfected cells with concentration range 0-150 nM, then observed fluorescence by fluorescence microscope to determine the optimal concentration.3.Different siRNA sequences transfected podocytes with optimal concentration,then used RT-qPCR to screen the optimal siRNA transfection sequence.4.MPC-5 were divided into normal control group、NOX4 siRNA group、negative siRNA group、HG group、HG+NOX4 siRNA group and HG+negative siRNA group,peak time point of NOX4 protein level as HG intervention time, then detected the NOX4 protein level by Western Blot and MVs of content by ELISA in each group.5.Statistical methods: used SPSS 18.0 statistical software for data analysising.The results were expressed as mean ± standard deviation( sx ?). More than two groups were compared by univariate analysis of variance(ANOVA). P<0.05 was considered statistically significant.Results:1. NOX4 mRNA test results: With prolongation of HG processed, the level of NOX4 mRNA was lower after the first rise trend, compared with normal control(NC)group, 2、4、6、12 and 24 hours was respectively 2.61、1.94、1.83、1.52、1.11 times of NC group, showing 2 hours reached a peak, then gradually decreased(P<0.01)。2. NOX4 protein test results: With prolongation HG processed, NOX4 protein level was lower after the first rise trend, 2、4、6、12 and 24 hours was respectively1.14、2.08、1.63、1.51、0.96 times of NC group, showing 4 hours reached a peak, then gradually decreased(P<0.05)。3. FAM-siRNA transfection results: green fluorescence was visible in cells by fluorescence microscope, the number of cells containing green fluorescence gradually increased with increase of siRNA concentration in 50-125 nM, when concentration was 150 nM, most cells were killed, suggesting 125 nM was the optimal concentration.4. NOX4 mRNA test results after different si RNA transfection: compared with normal control group, lipo2000 group, negative siRNA group, siRNA-A group and siRNA-C group, the level of NOX4 mRNA of siRNA-B group significantly decreased(P<0.05), suggesting si RNA-B was the optimal interference sequence.5. NOX4 protein level after HG stimulated cells of NOX si RNA transfection:compared with normal control group(group1), NOX4 protein level of NOX4 siRNA group(group2) was reduced by 54.1%(P<0.01); NOX4 protein level of HG group(group3) significantly increased compared with group 1,2 and was respectively2.20,4.79 times for group1,2(P<0.01); compared with group3, HG+NOX4 siRNA group(group4), NOX4 protein was reduced by 59.7%, and was 1.93 times of group 2(P<0.01);Negative siRNA group, HG+negative siRNA group, respectively, compared with group1, 3, there was no statistically significant difference(P>0.05).6. MVs content after HG stimulated cells of NOX siRNA transfection: compared with group1, MVs content of group2 was reduced by 17.6%, MVs content of group3 significantly increased, was respectively 2.1、2.55 times of group1、2(P< 0.01); MVs of group4 was reduced by 48.0% compared with group3(P<0.01),was increased by24.5% compared with group2(P<0.01), but no statistically significant difference with group1(P>0.05); Negative siRNA group, HG+negative siRNA group, respectively,compared with group1, 3, there was no statistically significant difference(P>0.05).Conclutions:HG can increase podocytes NOX4 mRNA and protein expression with time dependence.NOX4 siRNA interference can decrease MVs generation of podocytes with HG stimulating.HG may mainly by increasing NADPH oxidase NOX4 source of oxidative stress to promote production of MVs from podocytes.